PROJECT SUMMARY The intestinal epithelium is a critical mucosal barrier that mediates communication between the intestinal luminal environment and the immune system. Several lines of evidence suggest that stable reactive oxygen species (ROS) play an important role in regulating cell signaling at mucosal surfaces, both in response to the microbial environment and in gut inflammation. Stable ROS such as hydrogen peroxide (H2O2) can be generated by epithelial cells through the NADPH oxidase enzyme family (e.g. NOX1) or from other environmental sources such as bacteria. H2O2 modulates cellular signaling via oxidative modifications of target cysteine residues on proteins, altering downstream functions. However, exactly what signaling proteins and pathways are impacted by ROS during inflammation is poorly understood and may provide key information in understanding the pathogenesis of inflammatory bowel diseases and the development of biomarkers to track disease activity. Our recent data shows that NOX1-generated H2O2 can be transported through aquaporin channels (AQP3) and is important in mediating epithelial environmental responses. Our studies have also shown that epithelial signaling, in response to pattern-recognition-receptor ?danger? signals such as interleukin-1 beta (IL-1b), is also H2O2-dependent. To further investigate proteins that are selectively oxidized in the presence of IL-1b we carried out redox proteomics of mouse primary intestinal epithelial cells. We discovered several candidate proteins that are selectively oxidized in a NOX1 and AQP3-dependent manner, including Peroxiredoxin 6 (PRDX6) - a known multifunctional redox-sensitive protein. Aim 1 of this R03 proposal will therefore test the hypothesis that PRDX6 function is important for epithelial pattern-recognition-receptor signaling. Aim 2 takes a proteomics approach to identify key redox-modified proteins in human disease by investigating pattern-recognition-receptor responses using colonoids derived from healthy individuals and individuals with inflammatory bowel disease. The main goals of these studies are to identify novel redox- dependent signaling pathways in intestinal epithelial cells during inflammatory danger signaling and to use this data to develop novel therapeutic approaches for mucosal inflammatory diseases.